High CASC expression predicts poor prognosis of lung cancer: A systematic review with meta-analysis

Background The long non-coding RNA cancer susceptibility candidate (CASC) has abnormal expression in lung cancer tissues and may correlate with lung cancer prognosis. This study aimed to comprehensively evaluate the association between CASC expression and the cancer prognosis. Methods PubMed, Embase, Web of Science, Google Scholar, Cochrane Library, and China National Knowledge Infrastructure databases were searched until April 1, 2023, to obtain the relevant literature. Studies that met the predefined eligibility criteria were included, and their quality was independently assessed by 2 investigators according to the Newcastle-Ottawa Scale (NOS) score. Detailed information was obtained, such as first author, year of publication, and number of patients. Hazard ratio (HR) with a 95% confidence interval (CI) was extracted and grouped to assess the relationship between CASC expression and cancer prognosis. The dichotomous data was merged and shown as the odds ratio (OR) with a 95% CI was extracted to assess the relationship between CASC expression and clinicopathological parameters. Results A total of 12 studies with 746 patients with lung cancer were included in the meta-analysis. The expression levels of lncRNA CASC2 and CASC7 were decreased, while those of CASC9, 11, 15, and 19 were induced in lung cancer tissues compared with paracancerous tissues. In the population with low CASC expression (CASC2 and CASC7), high CASC expression indicated a good lung cancer prognosis (HR = 0.469; 95% CI, 0.271–0.668). Conversely, in the population with high CASC expression (CASC9, 11, 15, and 19), high CASC expression predicted a poor lung cancer outcome (HR = 1.910; 95% CI, 1.628–2.192). High CASC expression also predicted worse disease-free survival (DFS) (HR = 2.803; 95% CI, 1.804–6.319). Combined OR with 95% CI revealed an insignificant positive association between high CASC expression and advanced TNM stage (OR = 1.061; 95% CI, 0.775–1.454), LNM (OR = 0.962; 95% CI, 0.724–1.277), tumor size (OR = 0.942; 95% CI, 0.667–1.330), and histological grade (OR = 1.022; 95% CI, 0.689–1.517). Conclusion The CASC expression levels negatively correlate with lung cancer prognosis. Therefore, CASC expression may serve as a prognostic marker and a potential therapeutic target for lung cancer.


Introduction
Cancer is currently the top threat to global public health, imposing a heavy economic burden on the health system every year [1].According to the 2021 World Cancer Statistics Report, approximately 19.8 million new cancer cases are expected worldwide in 2020, with cancer deaths approaching 10 million [2].Compared with other cancers, lung cancer incidence and death toll rank first among men and third among women.The primary treatment for lung cancer is surgery combined with radiotherapy and chemotherapy [3,4] and can be combined with targeted therapy or immunotherapy to boost efficacy.Although these therapies increase the 5-year survival rate of the disease, its prognosis remains poor [5,6].Moreover, since some treatment methods have entered the bottleneck period, cancer research has refocused on discovering novel approaches using molecular biology [7].Many small molecules and compounds are involved in the occurrence and development of cancer and often investigated for finding new therapeutic targets and prognostic markers [8].
Long non-coding RNAs (lncRNAs) are associated with the occurrence and development of tumors [9].These RNAs are more than 200 nucleotides long, have no protein-coding ability, and regulate gene expression by directly acting on downstream genes or signaling pathways [10].They can also act as competitive endogenous RNAs that sponge and down-regulate microRNAs, indirectly regulating the expression of downstream targets [11] and affecting tumor cell proliferation, migration, invasion, and apoptosis [12,13].For example, Gu et al. reported that NNT antisense RNA 1 (NNT-AS1) may facilitate the proliferation, migration, and invasion of cholangiocarcinoma cells by enhancing epithelial-mesenchymal transition (EMT) via up-regulating E-cadherin and down-regulating N-cadherin [14].Chen et al. revealed that small nucleolar RNA host gene 8 (SNHG8) promotes the proliferation of nonsmall-cell lung cancer (NSCLC) cells by increasing the expression of cyclin D1 (CCND1) and cyclin dependent kinase 6 (CDK6) via sponging and down-regulating miR-542-3p [15].
Among lncRNAs, those from the lncRNA cancer susceptibility (CASC) gene family are typically deregulated in lung cancer [16].Abnormally expressed CASCs regulate the occurrence and development of the disease by affecting the proliferation, migration, invasion, and cisplatin resistance of lung cancer cells [17].Thus, mounting studies attempt to explore the correlation between CASC expression and lung cancer prognosis [18].However, because various CSACs have different expression levels in lung cancer, these studies are biased due to the small sample size, yielding contradictory results.Because of these shortcomings, a systematic review and meta-analysis were conducted in this study to determine whether a correlation exists between CASC expression and lung cancer prognosis.

Search strategy
In line with the reporting guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [19], 6 public electronic databases were browsed to obtain the suitable publications: PubMed, Embase, Web of Science, Google Scholar, Cochrane Library, and China National Knowledge Infrastructure.The related subject headings and search terms were as follows: "long non coding RNA cancer susceptibility," "lnc RNA cancer susceptibility," "cancer susceptibility," "lncRNA CASC," "lncCASC," or "CASC," and "Lung cancer," "NSCLC," "SCLC," or "Lung adenocarcinoma."The search also covered the references of the included articles.The search time was from the establishment of the database to April 1, 2023.

Inclusion and exclusion criteria
The included literature met the following criteria: 1.The research mainly explored the correlation between CASC expression and lung cancer prognosis.
2. Patients with lung cancer were divided into the CASC high expression group and low expression group based on RT-qPCR results.
3. A comprehensive detection method assessed the CASC expression levels in tumor tissue.
4. The study provided sufficient and usable data for evaluation.
The exclusion criteria were as follows: 1.The study data were insufficient or could not be evaluated.
2. The correlation between CASC expression and lung cancer prognosis was unevaluated.
3. The literature was a conference abstract, meta-analysis, or duplicate publication.
4. The study researched animals.

Quality assessment of included literature
Two researchers independently estimated the quality of included studies based on the Newcastle-Ottawa Scale (NOS) score.This scale evaluates primary studies through 3 modules and 8 items and involves the selection of the study population, comparability, and exposure/outcome evaluation [20].It adopts the semi-quantitative principle of the star system to assess the literature quality; the higher the score, the higher the quality of the research.Comparability was rated up to 2 stars, while the other items were up to 1 star.The full score was 9 stars.Studies with a score of no less than 6 were thought suitable for inclusion in the meta-analysis.

Extraction of relevant data
Two researchers independently extracted the data of the included studies as follows: first author, publication year, number of patients, cut-off value, survival outcome, and hazard ratio (HR) value with 95% confidence interval (CI).If a survival curve was provided without HR and its 95% CI, the Engauge Digitizer software (v.4.0) was employed to extract the HR value from the survival curve [21].In addition, clinicopathological parameters were obtained: TNM stage, lymph node metastasis (LNM), distance metastasis (DM), tumor size, histological grade, depth of invasion, and patient age and gender.If the extracted data were inconsistent, they were judged through discussion or consulting a third researcher.

Ethics approval
This was a clinical retrospective analysis and requires no ethical statement.

Statistical analysis
The RevMan software version 5.4.0 (Cochrane Collaboration, London, UK) and STATA version 12.0 (StataCorp LLC, College Station, TX) were utilized for statistical analyses.Hazard ratio values with 95% CIs were pooled to assess the relationship between CASC expression and survival prognosis of NSCLC.The dichotomous data were merged and presented as odds ratio (OR) and 95% CI to explore the association between CASC expression and the clinicopathological parameters.For the heterogeneity, a random-effect model was applied, and subgroup analysis was conducted for high heterogeneity (I 2 > 50, P < 0.05), while a fixed-effect model was adopted for low heterogeneity (I 2 � 50, P � 0.05).Results with P � 0.05 were considered statistically significant.The sensitivity analysis based on the STATA software was performed to explore the influence of data from individual studies on the overall results.The publication bias based on Begg's test was conducted to reveal potential bias. 3 Results

The basic characteristics of the included literature
Based on the subject headings, 6 electronic databases were searched exhaustively, and 342 original studies were initially obtained.Among them, 68 were excluded because of duplication and 241 due to an unevaluated correlation between CASC expression and lung cancer prognosis.

The association between CASC expression and survival outcome
Among the 12 studies included in the meta-analysis, 11 with 660 patients assessed the relationship between CASC expression and overall survival (OS) of patients with lung cancer.3).

The association between CASC expression and TNM stage
The meta-analysis investigated 8 studies with 507 patients to evaluate the association between CASC expression and the TNM stage.The combined OR with 95% CI predicted the insignificant positive relationship between high CASC expression and advanced TNM stage (OR = 1.061; 95% CI, 0.775-1.454)(Fig 3).Based on the CASC expression levels, the patients were assigned into a CASC low expression subgroup (CASC2 and CASC7) and a high expression subgroup (CASC9, 11, 15, and 19).In the high subgroup, increasing CASC expression indicated a poor lung cancer prognosis (OR = 1.745; 95% CI, 1.121-2.7189),whereas, in the low subgroup, rising CASC expression predicted a good prognosis (OR = 0.597; 95% CI, 0.371-0.960)(Table 4).

The association between CASC expression and other clinicopathological parameters
The relationship between CASC expression and other clinicopathological parameters was also explored: LNM, DM, tumor size, histological grade, depth of invasion, age, and gender.An  4).

Sensitivity analysis and publication bias
Finally, Begg's test was employed to conduct a sensitivity analysis and estimate publication bias.Removing the results of a single study would not significantly change the overall results,

Discussion
Long non-coding RNAs are small molecules without protein-coding ability and were once considered uninvolved in the biological behavior of cells [31,32].As the research on diseases gradually moved focus from the individual to genes, many lncRNAs were discovered to participate in the progression of many diseases, such as cancer [33], cardiovascular [34], metabolic, and neurodegenerative diseases.Moreover, numerous ncRNAs have abnormal expression in cancer cells, affecting events detrimental to cancer prognosis, such as proliferation, migration, invasion, and apoptosis.For example, Chen et al. reported that long intergenic non-protein coding RNA 1234 (LINC01234) promotes the proliferation and migration and inhibits the apoptosis of gastric cancer cells by increasing core-binding factor subunit beta (CBFB) expression via sponging and down-regulating miR-204-5p [35].Shi et al. demonstrated that long intergenic non-protein coding RNA 261 (LINC00261) hinders the proliferation, migration, and invasion of NSCLCs by suppressing the Wnt signaling pathway through sponging miR-522-3p [36].
The cancer susceptibility candidate family of lncRNAs contains dozens of cancer-related CASCs that promote or inhibit tumor progression by affecting cell proliferation, migration, invasion, and apoptosis [37].Many researchers have tried to explore the correlation between CASC expression and cancer patients.However, due to the insufficient sample size of a single study and inconsistent conclusions among different studies, the reliability and robustness of the results are elusive.Therefore, this meta-analysis study was performed to determine the correlation between CASC expression and the prognosis of lung cancer.
Although combined HR value and 95% CI showed that high CASC expression predicted poor prognosis of lung cancer, this result was not statistically significant.Because of the inconsistent expression of different CASCs in lung cancer, we conducted a subgroup analysis and found that high CASC expression predicts poor prognosis of the disease.Furthermore, since the statistical methods differ among the included original articles (univariate analysis vs. multivariate analysis), follow-up time, source of HR value (paper vs. survival curve), and cut-off value, a certain degree of bias was present in the overall results.To reduce the bias, we conducted a subgroup analysis and showed that, in univariate or multivariate analysis subgroups, highly expressed CASC predicted poor prognosis of lung cancer.High CASC expression also predicted poor lung cancer prognosis in the mean and median subgroups.In addition, we evaluated the correlation of CASC expression levels with clinicopathological parameters of the patients.We demonstrated that high CASC expression correlated with the advanced TNM stage, low lymph node and distant metastases, large tumor diameter, and low pathological grade, conforming CASC expression is associated with the proliferation, migration, and invasion of tumor cells.Although our data show high CASC levels predict poor lung cancer prognosis, the underlying molecular mechanism remains unclear.Nonetheless, its missing steps are slowly uncovered by recent evidence (Fig 8).Xiao et al. revealed that CASC2 contributed to the proliferation, migration, and invasion of cisplatin-resistant NSCLCs cells (A549/DDP) by up-regulating interferon regulatory factor 2 (IRF 2) via sponging and suppressing miR-18a [16] (Table 5).Similarly, Tong et al. revealed that CASC2c facilitates the invasion, proliferation, and migration of colorectal cancer cells by activating the β-catenin signaling pathways [17].He et al. demonstrated that CASC2 promotes the proliferation of NSCLCs but did not uncover the mechanism of this effect [22].Other CASC members also affect different aspects of growth and metastasis of lung cancer cells.For instance, Chen et al. showed that CASC7 inhibits the proliferation and migration of NSCLCs by down-regulating N-cadherin and vimentin but up-regulating E-cadherin expression via sponging miR-92a [23].Zhao et al. uncovered that CASC9 enhances the proliferation, migration, and invasion of NSCLCs by suppressing S100 calcium-binding protein A14 (S100A14) via sponging and down-regulating miR-335-3p [24].Moreover, Bing et al. reported that CASC9 contributes to the proliferation  This study has several shortcomings.The patients included in this study were Chinese, making the conclusions applicable only to China or Asia.In addition, the sample size included in this study was not large, rendering the results and conclusions prone to selection bias.Another drawback is that although some included studies specified the HR value and 95% CI, others only provided the survival curve, necessitating indirect calculation of HR and resulting in the overall HR value being prone to statistical bias.Most CASCs were highly expressed in lung cancer tissues, and high CASC expression predicted poor survival outcomes in these subgroups.However, some CASCs were down-regulated in lung cancer tissues, and low expression of these transcripts predicted poor survival outcomes in these subgroups.Therefore, subgroup analysis was performed based on the expression level of CASC in this study.Despite the above limitations, this study has many advantages.It is the first meta-analysis to explore the correlation between the expression level of the CASC family and lung cancer prognosis.It showed by summarizing a large amount of data that high CASC expression predicts poor survival outcomes in lung cancer with high reliability and robustness.This study comprehensively summarized all the possible molecular mechanisms by which CASC affects lung cancer progression, providing a valuable reference for cancer treatment.It also provided a detailed subgroup analysis, comprehensively presenting the prognostic correlation between CASC expression and different subgroups and between CASC expression levels and various clinicopathological parameters.

Conclusion
Most CASCs are highly expressed in lung cancer and predict its poor prognosis.Thus, CASCs are potential therapeutic targets and promising prognostic markers in lung cancer.

Table 1 . Basic features of the publications included in this meta-analysis (n = 12). author and year sample size lncRNA expression level cut-off value survival outcome HR with 95%CI analysis method source of HR value follow-up month NOS score
The combined HR with 95% CI showed an insignificant positive relation between elevated CASC expression and poor OS of patients with lung cancer (HR = 1.272; 95% CI, 0.686-1.858)(Fig2).High heterogeneity (I 2 = 85.8;P < 0.0001) was also discovered; in lung cancer tissues with Note: CASC, cancer susceptibility candidate; OS, overall survival; HR, hazard ratio; CI, confidence interval; NOS, Newcastle-Ottawa Scale.https://doi.org/10.1371/journal.pone.0292726.t001low CASC expression (CASC2 and CASC7), high expression of the transcript indicated a good prognosis of lung cancer.Conversely, in lung cancer tissues with high CASC expression (CASC9, 11, 15, and 19), high CASC expression predicted a poor prognosis.Based on the CASC expression levels in lung cancer tissues, a subgroup analysis was conducted, revealing that in the subgroup with high CASC expression, increasing transcript expression